1. Technical Field
The present invention relates to a method for calibrating an acoustic droplet dispensing apparatus, and more particularly to a method having a step in which the volume of a droplet is correlated to the liquid level of the source liquid.
2. Background Information
FIG. 1 illustrates an acoustic droplet dispensing apparatus 10 known in the prior art. Apparatuses of this type are capable of dispensing droplets of liquids having volumes as small as approximately one hundred picoliters, and are particularly useful in the biotechnology and biopharmaceutical fields. A representative acoustic droplet dispensing apparatus is described in U.S. Pat. No. 6,863,362 which is incorporated herein by reference.
In the apparatus 10, an acoustic wave emitter 14 (such as a piezoelectric crystal) is in electrical communication with a computer 18. During operation the acoustic wave emitter 14 generates an acoustic wave or beam 20 that can be propagated through an optional wave channel 24. The acoustic wave can be focused by a lens 28 prior to propagating through a coupling medium 32 to optimize the energy of the acoustic wave or beam 20 upon the liquid/air interface (free surface) of a source liquid 40. The assembly comprised of the acoustic wave emitter 14, the wave channel 24 and the lens 28 is referred to as an acoustic emitter assembly 29. The acoustic wave 20 is propagated through the coupling medium 32 after which the wave is transmitted through a source liquid containment structure 44 where the wave comes to focus at or near the surface of the pool of source liquid 40, thereby causing a droplet 60 of the source liquid 40 to be dispensed from the surface of the pool.
Examples of source liquid containment structures 44 include single and multi-well wellplates commonly used in molecular biology applications, capillaries (e.g., capillary arrays), and the like. However, other containers or structures may be used to hold the liquid 40 to be dispensed or ejected. A typical wellplate comprises a matrix (rows and columns) of individual wells 46. Typical commercially available wellplates have 96, 384, 1536 or 3456 individual wells. The source liquid 40 may be contained in some or all of these wells 46 and the composition of the source liquid in individual wells may differ from well to well (i.e. there can be multiple source liquids 40). Furthermore, the volume of source liquid in the individual wells may differ from well to well. The volume of source liquid in an individual well is derived from the liquid level and well geometry.
Optimally, in order to dispense one or more droplets from one of the individual wells 46, the well 46 must be positioned over the acoustic wave emitter 14. To accomplish this, the source fluid containment structure 44 is detachably affixed to a gripper 49. The gripper 49 is controlled by an actuator mechanism 50 which contains a horizontal actuator 54 for moving the containment structure 44 in the horizontal (x and y) directions. A vertical actuator 58 moves the acoustic wave emitter 14 and wave channel 24 in the vertical (z) direction. The actuator 50 is typically in communication with computer 18 which controls the movement of the containment structure 44 to select a source liquid 40 or to adjust focusing of the acoustic wave or beam 20 at or near the surface of the source liquid 40. The computer may have implemented thereon various algorithms to adjust the focal position and energy of the acoustic wave emitter as well as control and manage the location of the acoustic wave emitter relative to a particular source fluid present in or on a source fluid containment structure.
Accordingly, the apparatus 10 may be used to cause one or more droplets 60 of the source liquid 40 to be dispensed from the containment structure 44 and towards a target substrate 70, as is described in U.S. Pat. No. 6,863,362. The target substrate 70 may be a multi-well wellplate similar to the source fluid containment structure 44, or may be some other type of medium. Generally, one or more horizontal actuators 59 are provided for moving the target substrate 70 in the horizontal (x and y) directions. A typical wellplate that could be used as the target substrate 70 may have 96, 384, 1536 or 3456 individual target wells 74, or some other number of target wells. FIG. 2 illustrates the target wells 74 in a wellplate used as the target substrate 70.
In a preferred embodiment, a piezoelectric transducer is employed as an acoustic wave emitter 14. In one embodiment, a piezoelectric transducer comprises a flat thin piezoelectric element, which is constructed between a pair of thin film electrode plates. As is understood by those of skill in the art, when a high frequency and appropriate magnitude voltage is applied across the thin film electrode plates of a piezoelectric transducer, radio frequency energy will cause the piezoelectric element to be excited into a thickness mode oscillation. The resultant oscillation of the piezoelectric element generates a slightly diverging acoustic beam of acoustic waves. By directing the wave or beam onto an appropriate lens having a defined radius of curvature (e.g., a spherical lens, or the like), the acoustic beam can be brought to focus at a desired point.
In one embodiment, a computer sends an analog voltage pulse to the piezoelectric transducer by an electrical wire 78. The electronics can control the magnitude and duration of the analog voltage pulses, and also the frequency at which the pulses are sent to the piezoelectric transducer. Each voltage pulse causes the generation of an acoustic wave from the piezoelectric transducer, which in turn is propagated through a coupling medium and into or through the source fluid thereby impinging on the surface of the source fluid.
A problem that is encountered in using acoustic droplet dispensing systems, such as the apparatus 10, is that it is difficult to precisely control the volume of the droplets dispensed from the apparatus. In large part, this is because many parameters associated with the source liquid, such as chemical composition, viscosity, temperature, speed of sound in the liquid, etc., affect the size (volume) of the droplet. Furthermore, the liquid level of the source liquid in the well 46 also affects the size (volume) of the droplet. Additionally, other factors, such as the geometry of the source well (e.g. well shape, well bottom thickness, etc.) or the manufacturing variability of the acoustic emitter assembly 29, can influence the size of the droplet. Therefore, what is needed is a technique for calibrating acoustic droplet dispensing systems so that uniform droplet volume can be achieved.